Ku-70-derived Bax-suppressing peptides and use thereof for the protection of damaged cells

ABSTRACT

A method of protecting cells from cell death comprising the step of supplying to the cell an effective amount of a Bax-inhibiting peptide is disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional applicationNos. 60/360,755 and 60/384,204 and U.S. application Ser. No. 10/247,045.All three applications are incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTBACKGROUND OF THE INVENTION

[0002] Bcl-2 family proteins are known to regulate a distal step in anevolutionarily conserved pathway for programmed cell death, with somemembers functioning as suppressors of apoptosis and others as promotersof cell death (Gross, et al., Genes Dev. 13:1899-1911, 1999; Reed,Nature 387:773-776, 1997). In mammalian cells, Bcl-2 family proteins areknown to control mitochondria-dependent cell death cascades (Adams andCory, Science 281:1322-1326, 1998; Green and Reed, Science281:1309-1312, 1998; Reed, et al., Cancer J. Sci. Am. 4 Suppl. 1: S8-14,1998). Mitochondria release apoptogenic factors during apoptosis such ascytochrome c apoptosis-inducing factor (AIF), and SMAC/DIABLO (Green,2000). cytochrome c released from mitochondria into the cytosol spacetriggers Apaf-1-dependent caspase activation leading cells to death(Green, Cell 102:1-4, 2000; Zou, et al., Cell 90:405-413, 1997).Pro-apoptotic Bcl-2 family proteins such as Bax promote cytochrome crelease from mitochondria (Jurgensmeier, et al., Proc. Natl. Acad. Sci.USA 95:4997-5002, 1998). On the other hand, anti-apoptotic Bcl-2 familyproteins such as Bcl-2 suppress cytochrome c release from mitochondria,thereby protecting cells from apoptotic signals triggered by severalstimuli (Kluck, et al., Science 275:1132-1.136, 1997; Yang, et al.,Science 275:1129-1132, 1997). The relative ratios of these various pro-and anti-apoptotic members of the Bcl-2 family have been known todetermine the sensitivity of cells to diverse apoptotic stimuli (Oltvaiand Korsmeyer, Cell 79:189-192, 1994) including chemotherapeutic drugsand radiation, growth factor deprivation, loss of cell attachment toextracellular matrix, hypoxia (a common occurrence in the centers oflarge tumors), and lysis by cytotoxic T-cells (Adams and Cory, supra,1998; Green and Reed, supra, 1998; Gross, et al., supra, 1999; Reed,Semin. Hematol. 34:9-19, 1997).

[0003] Among pro-apoptotic Bcl-2 family members, Bax and Bak play a keyrole for apoptosis induction. The double knock out of these genes inmice resulted in the resistance of the cells to several cell deathstimuli known to trigger mitochondria-dependent apoptosis, such asUV-irradiation, staurosporin (pan-kinase inhibitor), and someanti-cancer drugs (Wei, et al., Science 292:727-730, 2001). Bax normallyresides in the cytosol in a quiescent state. Upon receipt of apoptoticstimuli, Bax translocates into mitochondria (Wolter, t al., J. Cell.Biol. 139:1281-1292, 1997), and promotes cytochrome c release, possiblyby forming a pore in the mitochondrial outer membrane (Korsmeyer, etal., Cell Death Differ. 7:1166-1173, 2000; Saito, et al., Nat. CellBiol. 2:553-555, 2000). On the other hand, anti-apoptotic familyproteins such as Bcl-2 and Bcl-XL reside in the mitochondrial membraneand antagonize the cytotoxic activity of Bax moved from the cytosol(Adams and Cory, supra, 1998; Green and Reed, supra, 1998; Reed, et al.,supra, 1998). Mitochondrial translocation of Bax is one of the criticalsteps for the induction of apoptosis, however the mechanism is not yetfully understood.

[0004] Translocation of Bax from the cytosol to the mitochondria iscaspase-independent, since caspase-inhibitor pretreatment does notinterfere with this process (Goping, et al., J. Cell Biol. 143:207-215,1998). C-terminus hydrophobic residues forming the ninth (x-helix of Baxare reported to be involved in the translocation of Bax to themitochondrial membrane (Suzuki, et al., Cell 103:645-654, 2000). On theother hand, the N-terminus of Bax functions as a cytosol retentiondomain, since the deletion of this region allowed Bax to accumulate inthe mitochondrial membrane in the absence of apoptotic stimuli (Goping,et al., supra, 1998). The previous observations suggest thatunidentified cytosolic factor(s) interact with the N-terminus of Bax toinhibit its translocation to mitochondria in the absence of an apoptoticstimulus.

[0005] U.S. application Ser. No. 10/247,045 describes the suppression ofBAX by Ku-70, a factor that binds the N-terminus of Bax and prevents itsmitochondrial translocation. The present invention involves thedevelopment of a membrane permeable peptide that inhibits Bax-mediatedapoptosis.

BRIEF SUMMARY OF THE INVENTION

[0006] In one embodiment, the present invention is a method ofprotecting cells from cell death comprising the step of supplying to thecell an effective amount of a composition comprising a Bax-inhibitingpeptide. In one specific embodiment, the peptide comprises a peptideselected from the group consisting of the VPMLK, PMLKE and PMLK.

[0007] In another embodiment, the peptide is of the following formula:X¹PX²LX³X⁴, wherein X¹ is selected from amino acids with non-polar sidechain; X² is selected from amino acids with non-polar side chain; X³ isselected from amino acids with charged polar side chain; X⁴ is selectedfrom amino acids with charged polar side chain; and either X¹ or X⁴ maybe absent, although both may not be absent.

[0008] In one preferred embodiment, the Bax-inhibiting peptide isadministered to a patient.

[0009] In one embodiment, the invention is a preparation of one of thecompositions comprising peptides described above.

[0010] In another embodiment, the invention is a method of protectingcells from cell death comprising the steps of: (a) designing acomposition comprising a peptide or chemical that mimics theBax-suppressing function of VPMLK, PMLKE or PMLK, and (b) supplyingcells with an effective amount of the composition.

[0011] In another embodiment, the invention is a pharmaceuticalcomposition comprising a Bax-inhibiting peptide and a pharmaceuticalcarrier, wherein the peptide is of the following formula: X¹PX²LX³X⁴,wherein X¹ is selected for amino acids with non-polar side chain; X² isselected for amino acids with non-polar side chain; X³ is selected foramino acids with charged polar side chain; X⁴ is selected for aminoacids with charged polar side chain; and either X¹ or X⁴ may be absent,but both may not be absent.

[0012] The invention is also a method of preserving cells and organs fortransfusions or transplantation comprising storing the cells or organsin an effective amount above-identified peptide.

[0013] The invention is also a method of regeneration of damaged cells,comprising storing the cells in an effective amount of the peptide.

[0014] The invention is also a method of improving transfectionefficiency of genes or proteins into cells, comprising storing the cellsin an effective amount of the peptide.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0015]FIG. 1 illustrates that new peptides designed from Ku70 showanti-Bax activity. FIG. 1A: scheme of Ku70 full-length (Ku70 wt),Ku70₁₋₅₇₇ (Ku70Δ578-609), or Ku70₅₇₈₋₆₀₉ (Ku70Δ1-577). FIG. 1B: HEK293Tcells (10⁶ cells) were transfected with 1.0 ug of pCMV-2B-control vector(Flag-Control), pCMV-2B-Ku70 wt (Flag-Ku70 wt), pCMV-2B-Ku70₁₋₅₇₇(Flag-Ku70₁₋₅₇₇), or pCMV-2B-Ku70₅₇₈₋₆₀₉ (Flag-Ku70₅₇₈₋₆₀₉). One dayfollowing transfection, co-immunoprecipitation of Flag-Ku70 and Bax wasperformed. FIG. 1C: HEK293T cells were transfected with 1.0 ug ofpCMV-2B-control vector (Control) or pcDNA3-Bax (Bax) together with 2.0ug of pCMV-2B-Ku70 wt (Ku70 wt), pCMV-2B-Ku70₁₋₅₇₇ (Ku70₁₋₅₇₇), orpCMV-2B-Ku70₅₇₈₋₆₀₉ (Ku70₅₇₈₋₆₀₉). All the cells were alsoco-transfected with 0.5 ug pEGFP for the marking of transfected cells.Apoptosis in the transfected cells was analyzed 24 hours followingtransfection with Hoechst dye staining of the nucleus as described inMethods. FIG. 1D: HEK293T cells were incubated with 200 uM peptidecomposed of Ku70₅₇₈₋₅₈₇ or Ku70₅₉₆₋₆₀₀ for 4 hours using BioPorterreagent, and then transfected with 1.0 ug of pCDNA3-control vector(Control) or pcDNA3-Bax (Bax). The number of apoptotic cells wasdetermined as described in FIG. 1C. FIG. 1E: HEK293T cells (10⁶ cells)were incubated with 200 uM IPMIK (negative control peptide) or VPMLK(V5) and PMLKE (P5) peptides at the indicated concentrations for 1 hourand then incubated cells were transfected with 1.0 ug of pcDNA3-Bax.Also, unincubated cells were transfected with 1.0 ug of pcDNA3-Bax inthe presence or absence of 200 uM z-VAD-fmk. The number of apoptoticcells was determined as described in FIG. 1C. FIG. 1F: Summary of theanti-Bax activity and sequence of all peptides used.

[0016] FIGS. 2A-L are graphs demonstrating inhibitory effects ofBax-inhibiting peptides (BIP) against various apoptotic stimuli. FIGS.2A and 2B: HeLa cells (10⁶ cells) were preincubated with 200 uM negativecontrol (NC) peptide for 1 hour or V5 peptide for the indicated periods,and then treated with 200 nM STS (A) or 1200 J/m² UVC-irradiation (B).After 24 hours of apoptotic treatment, apoptotic cells were counted asdescribed in FIG. 1. FIG. 2C: HeLa cells were preincubated with V5peptide and/or z-VAD-fmk (Calbiochem), a pan-caspase inhibitor, at theindicated concentrations for 1 hour and then exposed to 1200 J/m² ofUVC-irradiation. The number of apoptotic cells was determined one dayafter UVC-irradiation as described in FIG. 1. FIGS. 2D-L: U87-MG glioma(D-F), MCF-7 breast cancer (G-I), and LNCaP prostate cancer cells (J-L)were preincubated with 200 uM negative control (NC) peptide or V5peptide at the indicated concentrations, and then treated with 20 uMetoposide (D, G, J), 20 uM cisplatin (E, H, K), or 1 uM doxorubicin (F,I, L). Apoptotic cells were analyzed at the indicated periods followingthe treatment with anti-cancer drugs as described in FIG. 1.

[0017] FIGS. 3A-F are a set of graphs illustrating the effects of BIP inBax- or Ku70-deficient cells. FIGS. 3A-D: Du145 cells (10⁶ cells) weretransfected with 1.0 ug pcDNA3-control vector (Control) or 0.125 ugpcDNA3-Bax (Bax) together with 0.5 ug pEGFP for the marking oftransfected cells. One day following transfection, cells were incubatedwith 200 uM negative control (NC) peptide, V5 peptide, or P5 peptide for1 hour, and then treated with 200 nM STS (A, B) or 1200 J/m² ofUVC-irradiation (C, D). After 24 hours of apoptotic treatment, apoptoticcells were counted as described in FIG. 1. BIP does not require Ku70 tosuppress apoptosis (E, F). Mouse embryonic fibroblasts (MEF) derivedfrom Ku70-deficient (Ku70−/−) mice were treated with 200 nM STS (E) or1200 J/m² UVC-irradiation (F) in the presence of 200 uM negative control(NC) peptide or V5 peptide. Apoptotic cells were analyzed at theindicated periods following the treatment with STS or UVC-irradiation asdescribed in FIG. 1.

[0018] FIGS. 4A-F demonstrate that BIP inhibits the mitochondrialtranslocation of Bax (A, B). FIG. 4A: One day following UVC-irradiationor STS-treatment in the absence (UV and STS) or presence of 200 uMnegative control (NC) peptide (UV+NC and STS+NC) or V5 peptide (UV+V5and STS+V5), subcellular fractionation of HeLa cells (10⁶ cells) wasperformed. FoF1 ATP synthase subunit α (F1α) was used to mark themitochondrial fraction. HM stands for “Heavy Membrane” fractioncontaining mitochondria. FIG. 4B: HeLa cells (10⁶ cells) were treatedwith 200 nM STS in the presence of 200 uM negative control (NC) peptide(STS+NC) or V5 peptide (STS+V5). One day following STS-treatment,subcellular fractionation was performed and the levels of Ku70 and Baxwere examined. Lactate dehydrogenase (LDH), PCNA, and F1α were used asmarkers for cytosolic, nuclear, and mitochondrial fractions,respectively. FIG. 4C: cytochrome c release from mitochondria isinhibited by BIP, but not z-VAD-fmk. HeLa cells (10⁶ cells) were treatedwith 200 nM STS in the presence or absence of 200 uM negative control(NC) peptide, V5 peptide, or z-VAD-fmk for 24 hours. Cytochrome creleased from mitochondria into cytosol was analyzed by subcellularfractionation followed by Western blot analysis of cytochrome c (cyt c)as well as mitochondrial FoF1-ATP-synthase subunit F1α (F1α) asdescribed in Methods. FIG. 4D: BIP suppresses STS-induced Caspaseactivation as well as z-VAD-fmk. HeLa cells (10⁶ cells) were treatedwith 200 nM STS in the presence or absence of 200 uM negative control(NC) peptide, V5 peptide, or z-VAD-fmk for 24 hours. Caspase activitywas assessed as described in Methods. FIG. 4E: BIP dose-dependentlybinds to endogenous Bax. Co-precipitation of endogenous Bax and BIP.HEK293T cells (10⁷ cells) were lysed in CHAPS buffer in the absence (Nopeptide) or presence of 200 uM biotin-labeled negative control (NC)peptide and biotin-labeled V5 peptide at the indicated concentrations.Co-precipitation experiments were performed with streptavidin beads oranti-Bax polyclonal antibody using CHAPS buffer as described in Methods.Western blot analyses of pre-precipitation (Input) and precipitatedsamples (IP) were performed using an anti-Bax monoclonal antibody. FIG.4F: Scatchard analysis of the interaction of BIP and Bax. Scatchardanalysis of the binding of FITC-labeled BIP (VPMLK) and endogenous Baxin Ku70-deficient MEFs was performed as described in Methods. Thedissociation constant (Kd) was estimated to be 1.3 uM (1/Ka) for thisinteraction. No significant binding of FITC-BIP to the cellularcomponents was detected in Bax-immunodepleted cell lysates.

[0019] FIGS. 5A-J demonstrate intracellular distribution of membranepermeable BIP and co-localization with endogenous Bax. FIGS. 5A-B: HeLa(A) or Du145 cells (B) (10⁶ cells) were incubated with 200 uM V5 peptidelabeled with FITC for 1 hour and then pictures were taken under afluorescent microscope. Localization of FITC-BIP changes in accordancewith restoration of Bax protein in Bax-deficient Du145 cells. FIGS.5C-F: Bax-deficient Du145 cells (10⁶ cells) were transfected with 1.0 ugof pcDNA3-control vector (Du145Nector) or 1.0 ug pcDNA3-Bax (Du145/Bax)in the presence of 200 uM V5 peptide labeled with FITC. Twenty-fourhours later, cells were collected and the levels of Bax as well asβ-Tubulin were examined using total cell lysates (20 ug protein/lane)(F). Then the transfected cells were also immunostained by Texas-Redlabeled Bax as described in Methods and pictures were taken byfluorescent microscope (C-E). FIGS. 5G-I: HeLa cells were incubated with200 uM FITC-V5 peptide (G) for 1 hour, and then immunostained by Baxlabeled with Texas-Red (H). Pictures including a merged image (I) weretaken by fluorescent microscope. FIG. 5J: BIP suppresses the activationof Bax. HeLa cells (10⁶ cells) were treated with 1200 J/m²UVC-irradiation in the absence (UV) or presence of 200 uM negativecontrol (NC) peptide (UV+NC) or V5 peptide (UV+V5). One day afterUVC-irradiation, co-immunoprecipitation was performed with anti-Baxmonoclonal antibody (clone 6A7) using CHAPS buffer as described inMethods.

[0020] FIGS. 6A-D demonstrate that Ku70-derived peptides bind Bax anddissociate Ku70 from Bax. FIGS. 6A-D: HEK293T cells (10⁷ cells) lysed inCHAPS buffer were incubated with 200 uM negative control (NC) peptide or(A) VPMLK (V5), (B) PMLKE (P5), (C) PMLK (P4), and (D) MLKE (M4) at theindicated concentrations for 1 hour. Immunoprecipitation was performedwith anti-Ku70 monoclonal antibody or anti-Bax polyclonal antibody usingCHAPS buffer as described in Methods. Mouse IgG and pre-immune rabbitserum (NRS) were used as negative controls.

[0021]FIG. 7 demonstrates optimization of Bax-plasmid transfection intoDu145 cells. FIG. 7A: Bax-deficient Du145 cells (10⁶ cells) weretransfected with 1.0 ug of pcDNA3-control vector (Du145Nector) orpcDNA3-Bax (Du145/Bax) at the indicated concentrations. Twenty-fourhours later, cells were collected and the levels of Bax as well asβ-Tubulin were examined using total cell lysates (20 ug protein/lane).FIG. 7B: All the cells in FIG. 1A were also co-transfected with 0.5 ugpEGFP for the marking of transfected cells. Apoptosis in the transfectedcells was analyzed 24 hours following transfection with Hoechst dyestaining of the nucleus as described in Methods. The concentration of0.125 ug (10⁶ cells) was chosen to restore non-toxic levels of Bax inDu145 cells. FIG. 7C: BIP does not interfere the interaction ofKu70/Ku80. HEK293T cells (10⁷ cells) were lysed in CHAPS buffer andimmunoprecipitation was performed with anti-Ku80 mouse monoclonalantibody or anti-Ku70 rabbit polyclonal antibody in the presence(Anti-Ku80+BIP) or absence (Anti-Ku80) of 200 uM V5 peptide using CHAPSbuffer as described in Methods. Pre-immune rabbit serum (NRS) and mouseIgG were used as negative controls. Western blot analyses ofpre-immunoprecipitation (Input) and immunoprecipitated samples (IP) wereperformed by anti-Ku70 rabbit polyclonal antibody or anti-Ku80 mousemonoclonal antibody. FIG. 7D: BIP does not affect Bax/Bcl-2heterodimerization. HEK293T cells (10⁷ cells) were lysed in NP40 bufferand immunoprecipitation was performed with anti-Bax rabbit polyclonalantibody in the presence (Anti-Bax+BIP) or absence (Anti-Bax) of 200 uMV5 peptide using NP40 buffer as described in a previous report (Hsu andYoule, J. Biol. Chem. 23:10777-10783, 1998). Pre-immune rabbit serum(NRS) was used as a negative control. Western blot analyses ofpre-immunoprecipitation (Input) and immunoprecipitated samples (IP) wereperformed by anti-Bax mouse monoclonal antibody or anti-Bcl-2 mousemonoclonal antibody. FIG. 7E: BIP does not interact with Bak. HEK293Tcells (10⁷ cells) were lysed in CHAPS buffer in the presence of 200 uMbiotin-labeled negative control (NC) peptide or biotin-labeled V5peptide (BIP). Co-precipitation was performed with streptavidin beadsusing CHAPS buffer as described in Methods. Western blot analyses ofpre-precipitation (Input) and precipitated samples (IP) were performedby anti-Bax polyclonal antibody or anti-Bak polyclonal antibody.

[0022]FIG. 8 demonstrates that BIP inhibits Bax-mediated apoptosis asmeasured by propidium iodide (P1) exclusion. HEK293T cells (10⁶ cells)were transfected with 1.0 ug of pcDNA3 (Control) or pcDNA3-Bax (Bax) inthe absence or presence (Bax+BIP) of 200 uM V5 peptide. HBSS (Hanks'balanced salt solution)-washed live cells were incubated with 1 ug/ml ofP1 (Sigma) for 10 minutes at 4° C. in the dark. Flow cytometry wasperformed using a Becton Dickinson FACScan instrument. The percentageshown in the figure indicates the percentage of “dead” cells(PI-positive).

DETAILED DESCRIPTION OF THE INVENTION

[0023] In General

[0024] Bax is a pro-apoptotic member of Bcl-2 family of proteins andplays a key role in mitochondria-dependent apoptosis. Bax resides in thecytosol as a quiescent protein, and translocates into mitochondria uponthe receipt of apoptotic stimuli. Ku70 has been known to be a 70 kDasubunit of Ku-complex that plays an important role in DNA double strandbreak repair in the nucleus. We reported that Ku70 interacts withpro-apoptotic protein Bax in the cytosol and prevents the mitochondrialtranslocation of Bax, and thus Ku70 suppresses Bax-mediated apoptosis.(See U.S. Ser. No. 10/247,045)

[0025] In the present invention, we disclose the development of a newmembrane permeable peptide (Bax-Inhibiting Peptide; BIP) that inhibitsBax-mediated apoptosis and appears to mimic Ku70 in its interaction withthe Bax molecule. In one embodiment, BIP is comprised of five aminoacids designed from Bax-binding domain of Ku70 and suppresses themitochondrial translocation of Bax. A BIP inhibits Bax-mediatedapoptosis induced by saturosporin, UVC-irradiation, and anticancer drugsin several types of cells as disclosed below in the Examples.

[0026] By testing several deletion mutants of the Ku70 protein, I haveidentified the Bax-binding domain in Ku70. The domain comprises 6 aminoacids (VPMLKE). This peptide (Ku70 Peptide V6) inhibits the interactionof Ku70 and Bax at the concentration of 20-80 μM in lysates preparedfrom human cultured cells (HeLa cells and human kidney epithelial 293cells). Negative control experiments using the scrambled sequence ofthese 6 amino acids and the immediate next six amino acid sequence ofKu70 (Ku70 573-578 peptide, termed “Ku70 Peptide NC”) did not affect theinteraction of Ku70 and Bax, indicating the specificity of Ku70 PeptideV6 activity. Delivering the Ku70 peptide V6 into the cells also inhibitsmitochondrial translocation of Bax in the cells treated by severalapoptotic stresses such as UV-irradiation and staurosporin-treatment.

[0027] Ku70 Peptide V6 also suppressed cell death of human culturedcells (HeLa cells) treated by UV-irradiation and staurosporin. VPMLK(V5) and PMLKE (P5), deletion mutations, also showed anti-cell deathactivity. Importantly, V5 and P5 are membrane permeable and do notrequire a cell delivery system such as liposomes as in the case of V6.In one embodiment, the present invention is a Bax-inhibiting peptide,preferably VPMLKE, VPMLK, and PMLKE (V6, V5, and P5, respectively), thatcan protect cells from death and use of the peptide for this purpose.The sequences of these peptides were designed from the Bax-bindingdomain in the Ku70 protein (amino acid 578-583).

[0028] The original 6 amino acid peptide V6 (VPMLKE) and its variants,including shorter amino acid peptides (e.g. VPMLK (V5), PMLKE (P5)) andmodified peptides (e.g. modified for better membrane permeablization orlonger stability) are also Bax-inhibiting peptides, may be also suitabledrugs to protect cells and tissues from pathological damage and areincluded within the present invention.

[0029] The present invention also includes peptides (preferably 6-3residues) and chemicals (natural and synthetic compounds) designed tomimic the described Ku70 peptides. By “mimic,” I mean that the peptidehas at least 90% of the Bax-suppressing function of V5 and P5, asmeasured by the method of the Examples below. If a peptide or compoundsuppresses apoptosis by blocking the mitochondrial translocation of Bax,these chemicals or peptides successfully “mimic” Ku70 peptide.

[0030] Suitable methods for creating mimics can be found at: WO00/21980,EP1077218A2, WO01/60844, WO01/14412, WO01/55091, WO01/46197, WO02/20033,WO02/20034, WO02/20557, incorporated by reference. The followingarticles, incorporated by reference, would also guide one to make a Ku70mimic: P.C.A. Kam, “Platelet glycoprotein IIb/IIIa antagonists,”Anesthesiology 96:1237-1249, 2002; S. Mousa, “Antiplatelet therapies:From aspirin to GPIIb/IIIa inhibitors and beyond,” Drug Discovery Today4:552-561, 1999; R. S. McDowell, et al., “From peptide to non-peptide.2. The de novo design of potent, non-peptidal inhibitors of plateletaggregation based on a benzodiazepine scaffold,” J. Am. Chem. Soc.116:5077-5083, 1994; and B.K. Blackburn, et al., “From peptide tonon-peptide. 3. Atropisomeric GPIIb/IIIa antagonists containing the3,4-dihydro-1,4-benzodiazepine-2,5-dione nucleus,” J. Medicinal Chem.40:717-729, 1997.

[0031] Peptides with slight modifications (e.g., substitution of similarcharged amino acids or addition of 1, 2 or 3 innocuous amino acids ateither end or by the addition of an innocuous entity or moiety) to thepeptide sequences described herein are envisioned to be suitable BIPs.By “innocuous” I mean amino acid(s) or entities that do notsubstantially reduce the Bax-inhibiting activity of the core peptidesequence PMLK. Therefore, a composition comprising a Bax-inhibitingpeptide of the present invention includes a peptide described herein(e.g., PMLK, PMLKE, VPMLK, VPMLKE and the formula below) with additionsof 1, 2 or 3 innocuous amino acids at either end, innocuous amino acidsubstitutions, addition of innocuous moieties or entities, and mimics ofthese peptides.

[0032] Peptide drug delivery and therapeutic administration is limitedby permeability and selectivity problems involving the cell membrane(Morris, et al., Nat. Biotechnol. 19(12):1173-1176, 2001). Strategies todeliver peptides and proteins into cells may solve these problems. Manysmall protein domains, called protein transduction domains (PTD's), havebeen shown to cross biological membranes and act independently fromtransporters or specific receptors to promote delivery of peptides andproteins into cells. The work of Hawiger (Hawiger, Curr. Opin. Chem.Biol. 3(1):88-94, 1999) is one example of how we envision this techniqueinvolving peptide modification could be applied to create a compositioncomprising BIP that consists of adding a PTD domain at position X¹ or X⁴to aid in either the transport or BIP to specific target cells or to aidthe stability of the molecule.

[0033] The present invention also includes peptides in which sequencesdescribed above are repeated multiple times.

[0034] Because the amino acid sequences VPMLK and PMLKE are equallyeffective to suppress Bax, the amino acid sequence PMLK is considered tobe the core structure for BIP's biological activity. Indeed, PMLK issufficient to bind Bax in vitro. However, PMLK is not biologicallyactive because these four amino acids are not retained in the cell.Addition of V before P, or E after K of PMLK causes the peptide(s) to beeffectively retained inside the cells. Therefore, these peptides (VPMLKor PMLKE) express anti-Bax activity in cells and are Bax-inhibitingpeptides.

[0035] I assume that the addition of the fifth amino acid to PMLK isrequired for either solubility of BIP in the cytosol or protection ofthe export of BIP through cell membrane. Therefore, other amino acidswhich retain similar polarity are expected to be suitable substitutesfor V and E.

[0036] In PMLK, P and L seem to be required for effectiveness. Because Phas very unique structure among amino acids, and substitution of L withI (L and I are non-polar amino acids) diminish BIP's biological activity(described in Nature Cell Biology BIP paper).

[0037] In PMLK, M and K may be interchangeable with other amino acids inthe same group with similar polarity.

[0038] Based on the above logic, we describe the formula of the futuremodification of a preferred embodiment of the BIP as comprising thepeptide X¹PX²LX³X⁴, wherein

[0039] X¹=Amino acids with non-polar side chain, such as Glycine (G),Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M),Proline (P), Phenylalanine (F), Tryptophan (W).

[0040] X²=Amino acids with non-polar side chain, such as Glycine (G),Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Methionine (M),Proline (P), Phenylalanine (F), Tryptophan (W).

[0041] X³=Amino acids with charged polar side chain, such as Lysine (K),Arginine (R), Histidine (H), Aspartic acid (D), Glutamic acid (E), and

[0042] X⁴=Amino acids with charged polar side chain, such as Lysine (K),Arginine (R), Histidine (H), Aspartic acid (D), Glutamic acid (E).

[0043] Either X¹ or X⁴ may be absent.

[0044] I envision that 1, 2 or 3 innocuous amino acids or innocuousentities or moieties may be added at either end of the peptide or to thepeptide itself without significantly reducing its Bax-inhibitingactivity. These are suitable compositions comprising a Bax-inhibitingpeptide.

[0045] In a preferred embodiment, the present invention is apharmaceutical preparation comprising a BIP and a pharmaceuticalcarrier. The potential application of the drugs or pharmaceuticalpreparation based on this discovery are drugs to protect the death ofcells and tissues damaged by stroke, heart attack, ischemia,degenerative diseases (neuron and muscle, e.g. Alzheimer disease,Parkinson's disease, cardiomyocyte degeneration, etc), infection byparasitic organisms (virus, bacteria, yeast, or protozoa, etc),side-effects of other drugs (e.g. anti-cancer drugs), UV/X-rayirradiation, and several other pathological conditions triggering celldeath signals. Other potential applications include supporting theregeneration of damaged cells, including neuron and muscle cells;improving transfection efficiency of genes and proteins into cells, andpreserving cells and organs for transfusion or transplantation.

[0046] The following references describe the Bax protein playing a keyrole in various diseases: Injury-induced neuron death—Deckwerth, et al.Neuron. 17:401-411, 1996; Martin, et al., J. Comp. Neurol. 433:299-311,2001; Kirkland, et al., J. Neurosci. 22:6480-90, 2002; Alzheimerdisease—MacGibbon, et al., Brain Res. 750:223-234, 1997; Selznick, etal., J. Neuropathol. Exp. Neurol. 59:271-279, 2000; Cao, et al., J.Cereb. Blood Flow Metab. 21 :321-333, 2001; Zhang, et al., J. Cell Biol.156:519-529, 2002; Ischemia-induced cell damage—Kaneda, et al., BrainRes. 815:11-20, 1999; Gibson, et al., Mol. Med. 7:644-655, 2001; HIV(AIDS) and Bax: Castedo, et al., J. Exp. Med. 194:1097-1110, 2001;Drug-induced neuron death—Dargusch, et al., J. Neurochem. 76:295-301,2001; Parkinson's disease—Ploix and Spier, Trends Neurosci. 24:255,2001; Huntington's disease—Antonawich, et al., Brain Res. Bull.57:647-649, 2002.

[0047] One would most likely administer the BIP orally, by intravenousinfusion, intramuscular or subcutaneous injection, or by inhalation orintracranial injection.

EXAMPLES

[0048] Anti-Bax activity of new peptides designed from Ku70. We foundthat the C-terminal 74 amino acids of Ku70 are sufficient to bind Baxand to inhibit Bax-mediated apoptosis in several types of cells. Recentstructural analysis of Ku70 revealed that the 74 amino acid C-terminalportion is comprised of three α-helices and four flexible loop domains(FIG. 1A). Further analysis of a series of deletion mutants of Ku70revealed that the C-terminal 32 amino acids are sufficient for theinhibition of Bax-induced apoptosis in HEK293T cells (FIGS. 1A-C).Flag-tagged Ku70 mutant expressing the amino acids 578-609 (Ku70₅₇₈₋₆₀₉)of Ku70 binds endogenous Bax, whereas the Ku70 mutant deleted with theseamino acids (Ku70₁₋₅₇₇) did not (FIG. 1B). Ku70₅₇₈₋₆₀₉, but notKu70₁₋₅₇₇, suppressed Bax-induced apoptosis in HEK293T cells (FIG. 1C).These results suggest that the Bax-inhibiting domain of Ku70 localizesin the amino acids 578-609 of Ku70.

[0049] There are two α-helices in these 32 amino acids according to theprevious reports (FIG. 1A). Synthetic peptides corresponding to thesetwo α-helices were made and their activities to suppress Bax-inducedapoptosis were tested (FIG. 1D). Since these peptides are not membranepermeable, FITC-labeled peptides were delivered into the cells byliposome (BioPorter) and the presence of the peptides in the cells wereconfirmed by FITC fluorescence (not shown). The peptide of 578-587, butnot 596-600, inhibited Bax-induced apoptosis, suggesting that theBax-inhibiting domain is the 2^(nd) α-helix from the C-terminus of Ku70(FIG. 1D).

[0050] Further analysis of the peptides designed from Bax-inhibitingdomain in Ku70 revealed that the six amino acids (VPMLKE; V6-peptide) inthe Bax-inhibiting domain (amino acids 578-587) of Ku70 are sufficientto suppress Bax-mediated apoptosis (Bax-overexpression-, staurosporin(STS)-, or UVC-induced apoptosis) (data not shown). However, V6-peptideis not membrane permeable and liposome-mediated delivery of the peptideis required to suppress Bax-mediated apoptosis. Interestingly, thedeletion of one amino acid from VPMLKE at either the N-terminus or theC-terminus makes these peptides membrane-permeable, and did not abrogateBax-inhibiting activity (FIGS. 1E, F). As shown in FIGS. 1E, 1F and FIG.8, the five amino acids peptides VPMLK (V5) and PMLKE (P5) are equallyeffective in suppressing apoptosis induced by Bax. Since V5 or P5 ismembrane permeable, the addition of these peptides into the culturemedium is sufficient to block Bax-mediated apoptosis. However, furtherdeletion of one amino acid resulted in the abrogation of Bax-inhibitingactivity (FIG. 1F). When the amino acids V and L in V5-peptide (VPMLK)were changed with another non-polar amino acid 1, the peptide (IPMIK)lost its activity to suppress apoptosis (FIGS. 1E, F). The mutantpeptide IPMIK is used as negative control (NC) peptide in the followingexperiments.

[0051] BIPs inhibit various apoptotic stimuli mediated by Bax. BIPsshowed anti-apoptotic activities at the concentration of 50 uM-200 uM(FIG. 1E), as well as z-VAD-fmk (Caspase inhibitor) does. Pretreatmentof the cells with BIPs for 1 hour was sufficient to protect them fromSTS- and UVC-induced apoptosis in HeLa cells (FIGS. 2A, B). To be noted,BIPs retained the ability to suppress apoptosis for three days in theculture medium (FIGS. 2A, B). As shown in FIG. 2C, BIP did not enhancez-VAD-fmk's suppression of apoptosis induced by UVC-irradiation as wellas STS (not shown). Since BIP inhibits Bax upstream of caspaseactivation, BIP may not be able to exert an effect additive to that of acaspase-inhibitor. BIPs also suppressed apoptosis induced by anti-cancerdrugs (etoposide, cisplatin, and doxorubicin) in cancer cell linesincluding breast cancer cells (MCF-7), glioma cells (U87-MG), andprostate cancer cells (LNCaP) (FIGS. 2D-L). To confirm that BIPsuppresses Bax-mediated apoptosis, the effects of BIPs in Bax-deficientcells (Du145) were examined (FIGS. 3A-D and FIGS. 7A-B). BIPs did notsuppress STS- and UVC-induced apoptosis in Bax-deficient cells, whereasBIP showed anti-apoptotic activity in these cells when Bax expressionwas restored by plasmid transfection (FIGS. 3A-D). These results suggestthat BIP specifically inhibits a Bax-mediated signal in apoptosis. Theseresults are consistent with the observations that Ku70 did not showcytoprotection against STS and UVC-irradiation in Bax-deficient cells,and that Ku70 did not block Bak-induced apoptosis. Similarly, BIPs didnot suppress Fas- and TRAIL-induced apoptosis (not shown). Fas and TRAILcan trigger a mitochondria-independent cell death pathway, therefore,BIP may not be able to suppress apoptosis induced by these factors.These results suggest that BIP suppresses only Bax-mediated apoptosis.On the other hand, BIP suppressed STS- and UVC-induced apoptosis inKu70-deficient mouse embryonic fibroblasts (MEFs), suggesting that BIPdoes not require endogenous Ku70 to suppress Bax-mediated apoptosis(FIGS. 3E, F).

[0052] BIP interacts with Bax, and inhibits the mitochondrialtranslocation of Bax. BIP suppressed the mitochondrial translocation ofBax stimulated by STS and UVC-irradiation (FIG. 4A), as well as Ku70protein did. As reported in another article, apoptotic stimuli decreasedKu70 levels in the cytosol. BIP did not inhibit this Ku70 disappearance(FIG. 4B), suggesting that the anti-apoptotic activity of BIP is not dueto the inhibition of the degradation of endogenous Ku70. The release ofcytochrome c from mitochondria and Caspase activation triggered byapoptotic stimuli were also significantly suppressed by BIP (FIGS. 4C,D), further supporting that BIP protects cells by inhibitingBax-mediated mitochondria-dependent apoptosis pathway. The interactionof BIP and Bax was confirmed by experiments using the system ofbiotin-labeled BIP and streptavidin beads. Biotin-labeled BIP(biotin-VPMLK), but not negative control peptide (biotin-IPMIK)precipitated Bax from the cell lysates when the streptavidin beads wereadded to the samples (FIG. 4E). On the other hand, Bak was notprecipitated by biotin-VPMLK (FIG. 7E), indicating that BIP bindsspecifically to Bax, but not Bak. The reason why IPMIK (negative control(NC) peptide in the figures) did not suppress Bax-mediated cell death isthat this modified peptide fails to interact with Bax. The value of Kdfor the interaction of BIP and Bax was 1.3 uM when Scatchard analysiswas performed using FITC-labeled BIP (VPMLK) and endogenous Bax inKu70-deficeint MEF cell lysates (FIG. 4F).

[0053] The cells treated with FITC-labeled BIP (VPMLK) are shown inFIGS. 5A and B. FITC-BIP localized mostly in the cytosol in HeLa cells(FIG. 5A), but distributed in both the cytosol and the nucleus inBax-deficient Du145 cells (FIG. 5B). Bax expression in Du145 cellsincreased the cytosolic FITC-BIP (FIGS. 5C-F), suggesting that BIP bindsthe cytosolic Bax. Co-localization of Bax and BIP was also observed(FIGS. 5G-I). These results further support that BIP inhibitsBax-mediated apoptosis by interacting with Bax in the cytosol. It issuggested that the conformational change (the N-terminus exposure) ofBax occurs before its mitochondrial translocation and that Ku70 inhibitsthis event. The monoclonal antibody clone 6A7 is known to detectexposure of the N-terminus of Bax. BIP reduced the amount of Bax proteinrecognized by 6A7 antibody in the cells treated by apoptotic stimuli(FIG. 5J), suggesting that BIP inhibits the conformational change of Baxin the same way as Ku70.

[0054] Inhibition of the interaction between endogenous Ku70 and Bax bypeptides. BIP (VPMLK (V5) and PMLKE (P5)) inhibited the interaction(co-immunoprecipitation) of the endogenous Bax and Ku70 in a dosedependent manner (FIGS. 6A, B). On the other hand, BIP did not interferewith the interaction of Ku70/Ku80 or Bax/Bcl-2 heterodimerization (FIGS.7A-B). These results suggest that BIP specifically interacts with Baxthrough the Ku70-binding domain in Bax, and that BIP competes with Ku70to bind Bax. Interestingly, PMLK (P4) inhibited the interaction of Baxand Ku70 when it was added into the cell lysates (FIG. 6C), althoughPMLK (P4) could not suppress apoptosis in the cell culture. On the otherhand, MLKE (M4) did not interfere with Bax-Ku70 interaction (FIG. 6D).These results suggest that four amino acids of PMLK may be sufficient tobind Bax at least in cell lysate. As shown in FIG. 1, VPMLK and PMLKEwere able to suppress Bax-induced apoptosis. Although further analysisis required, failure of PMLK to suppress apoptosis in the culture may bethe result of rapid degradation, modification, or escape of this peptidein the living cells. It is also possible that addition of V (N-terminus)or E (C-terminus) to PMLK may improve the membrane permeability or thecytosolic retention of the peptide. Future biochemical study may answerthis question.

[0055] In summary, we have identified membrane permeable peptides (BIPs)derived from the Bax-binding domain of Ku70 that inhibit Bax-mediatedapoptosis. We also found that BIP inhibits exposure of the N-terminus ofBax induced by apoptotic stimuli. The mechanism of the activationprocess of Bax is still enigmatic. BIP may become a new tool toelucidate the mechanism of the conformational change of Bax. At present,it is unclear whether BIP can bind Bax directly. The possibility remainsthat other factors are involved in this interaction. Future biochemicalstudy analyzing the complex formation of purified inactive Bax proteinsand peptides may answer these questions. Bax-mediated cell demise isknown to be involved in several types of degenerative diseases and inloss of viability of the cultured cells (Wolter, et al., J. Cell Biol.139:1281-1292, 1997; Saito, et al., Nat. Cell Biol. 2:553-555, 2000;Korsmeyer, et al., Cell Death Differ. 7:1166-1173, 2000). The membranepermeable Bax-inhibiting peptides developed in this study may provideinformation leading to development of new therapeutics that act byregulating apoptosis.

[0056] Methods

[0057] Cell Culture and Apoptosis Detection

[0058] HEK293T and HeLa cells were cultured in MEM supplemented with 10%fetal bovine serum (FBS) and Du145 cells and mouse embryonic fibroblasts(MEFs) were in DMEM with 10% FBS. Transfection of the plasmids wasperformed by SuperFect (Qiagen) according to the manufacturer's manual.Apoptosis was induced by pcDNA3-human Bax (Bax-encodingplasmid)-transfection, Staurosporin (STS)treatment and UVC-irradiation.The amount of the plasmids, the concentration of STS, and the energy ofUVC-irradiation are described in the figure legends. Apoptosis in thetransfected cells was analyzed as follows: A plasmid encoding enhancedgreen fluorescent protein (EGFP) (0.5 ug to 10⁶ cells) was transfectedto all the groups to mark the transfected cells. After the treatment,cells were stained with Hoechst dye and cells with apoptotic nuclei werecounted in GFP-expressing cells under the fluorescent microscope asdescribed by Matsuyama, et al., J. Biol. Chem. 273:30995-31001, 1998.Each point in the figures showing percentages of apoptosis representsthe mean +/−SE of three experiments. Caspase activities of cells weremeasured by detecting the cleavage of fluorogenic substrate of Caspase(DEVD-afc) as previously described.

[0059] Plasmids

[0060] The plasmid pcDNA3-Bax (human) has been described. (Xu and Reed,Mol. Cell 1:337-346, 1998). The plasmid vectors pCMV-2B and pEGFP werepurchased from Stratagene and Clonetech, respectively, and humanfull-length Ku70 and the deletion mutants of Ku70 were subcloned intoBamH1 and Sal1 sites of pCMV-2B vector, and the deletion mutants of Baxwere subcloned into EcoR1 and Xho1 sites of pEGFP plasmid. Thefull-length Ku70 cDNA was prepared by RT-PCR using HeLa cell cDNA. Themutant constructs of Ku70 described in this article were prepared by2^(nd) step PCR mutagenesis method (Xu and Reed, supra, 1998).

[0061] Delivery of Peptide into the Cells

[0062] For the membrane permeable peptide, the stock solution wasprepared with PBS, and the peptides were directly added into medium andincubated for 1 hour or for the periods of time indicated in the figurelegends (FIGS. 2A, B) at 37° C. before apoptosis induction. Non-membranepermeable peptide was delivered using BioPorter reagent (Gene TherapySystems) according to the manufacturer's manual. BioPorter-based peptidedelivery was performed 4 hours before apoptosis induction.

[0063] Cytochrome c Detection

[0064] One day following the transfection of the plasmids or thetreatment of the cells with STS or UVC-irradiation, cells werere-suspended in 200 ul of homogenization buffer (250 mM Sucrose, 20 mMHEPES, pH 7.5, 10 mM KCI, 1.5 mM MgCl₂, 1 mM EDTA, 0.1 mMphenylmethylsulfonyl fluoride), and separation of the cytosol and heavymembrane fraction (containing mitochondria and ER) were performed aspreviously reported^(14, 15). Cytosolic fraction of 20 ug protein and 1ul of membrane fraction (out of total 50 ul) were analyzed byWestern-blot with cytochrome c antibody (BD-Pharmingen dilution 1:1000).

[0065] Immunoprecipitation

[0066] Endogenous protein: HEK293T (10⁷ cells) were lysed in 200 ul of“CHAPS-based buffer” (150 mM NaCl, 10 mM Hepes, pH 7.4, 1.0% CHAPS)containing protease inhibitors (100 times dilution of ProteaseInhibitors Cocktail; Sigma) according to the previously reported method(Hsu and Youle, J. Biol. Chem. 273:10777-10783, 1998). After precleaningof 600 ul of the sample with 50 ul of Protein G-Sepharose at 4° C. for 1hour, immunoprecipitations were performed by incubating 200 ul oflysates with 20 ul of Protein G-Sepharose preabsorbed with 2 ug ofanti-Bax polyclonal (BD-Pharmingen) or anti-Ku70 monoclonal antibody(BD-Pharmingen) at 4° C. for 2 hours. In some cases, 2 ug of anti-Baxmonoclonal antibody (clone 6A7, BD Pharmingen) was used to detect activeform of Bax. After extensive washing in the buffer, beads were boiled in40 ul of Laemmli buffer and 20 ul of the eluted proteins were subjectedto SDS-PAGE. Co-precipitation of biotin-labeled peptides and endogenousBax using streptavidin beads: HEK293T cells (10⁷ cells) lysed in 200 ulCHAPS-based buffer were incubated with biotin-labeled negative control(NC) peptide or VPMLK (V5) peptide for 1 hour. The concentrations of NCand V5 peptide labeled with biotin are described in the figure legends.Coprecipitation was performed with streptavidin beads prepared in aratio of 75% Streptavidin Sepharose to 25% CHAPS buffer according to themanufacturer's manual (Amersham Pharmacia Biotech). Flag-tagged-Ku70 andendogenous Bax: HEK293T cells (10⁶ cells) were co-transfected with 1.0ug of pcDNA3-Bax and 1.0 ug of pCMV-2B-control vector (Flag-taggedfirefly luciferase), pCMV-2B-Ku70 wt (Flag-Ku70 wt), pCMV-2B-Ku70₁₋₅₇₇(Flag-Ku70₁₋₅₇₇), or pCMV-2B-Ku70₅₇₈₋₆₀₉(Flag-Ku70₅₇₈₋₆₀₉) in thepresence of 50 uM z-VAD-fmk. Co-immunoprecipitation was performed withanti-Flag monoclonal antibody (Stratagene; 2 ug for 200 ul sample), andWestern-blot of Bax (15% SDS-PAGE) was done with anti-human Baxpolyclonal antibody (BD-Phramingen).

[0067] Subcellular Fractionation

[0068] One day after the treatment, cells were homogenized (Teflonhomogenizer) with 200 ul of ice-cold homogenization buffer (250 mMSucrose, 20 mM HEPES, pH 7.5, 10 mM KCI, 1.5 mM MgCl₂, 1 mM EDTA, 0.1 mMphenylmethylsulfonyl fluoride). Subcellular fractionation was performedas reported (Hoetelmans, et al., Cell Death Differ. 7:384-392, 2000),together with the confirmation of each fraction with appropriate markerproteins (cytosolic fraction; Lactate dehydrogenase (LDH) by anti-humanLDH antibody (Sigma), nucleus fraction; PCNA by anti-PCNA antibody(Oncogene), mitochondria containing heavy membrane fraction; F1-ATPaseα-subunit by anti-F1α subunit antibody (Molecular Probe). For total celllysates, samples were prepared with ice-cold lysis buffer (containing 50mM NaCl, 25 mM Hepes (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 ug/ulE-64, and 1% Triton X-100). In some experiments, samples containing 20ug of protein from total cell lysates or cytosolic fraction weresubjected to Western analysis of Bax or Ku70. The pellets of thefractions of heavy membrane and nucleus were dissolved in 50 ul ofLaemmli buffer and the same proportion of the volume equal to that ofthe cytosol samples out of its total volume was used for Western-blotanalysis.

[0069] Fluorescent Microscope Image

[0070] HeLa cells and transfected or untransfected Du145 cells withpcDNA3-Bax were incubated with 200 uM FITC-labeled VPMLK (V5) peptidefor 1 hour prior to microscopic analysis by fluorescent microscope. Forimmunostaining, cells incubated with FITC-V5 peptide for 1 hour werefixed with 4% paraformaldehyde in phosphate buffered saline (PBS). Cellswere permeablized with 0.5% Triton X-100 and 0.05% Tween-20 in PBS.Fixed cells were incubated in PBS containing 5% BSA at 37 C, and stainedwith anti-Bax monoclonal antibody (BD-Pharmingen, dilution 1:50)followed by the detection of Texas-Red labeled anti-mouse IgG (JacksonImmunoResearch, dilution 1:100).

[0071] Scatchard Analysis of the Binding of FITC-V5 and Bax

[0072] Ku70-deficient MEFs (10⁷ cells) were lysed in 200 ul of“detergent-free hypotonic buffer” (hypotonic (5 mM NaCl) phosphatebuffered saline, pH 7.4) containing protease inhibitors (100 timesdilution of Protease Inhibitors Cocktail; Sigma) according to thepreviously reported method (Hsu and Youle, supra, 1998). For Scatchardanalysis, the cytosol fraction was used and NaCl was added to preparethe isotonic condition before immunoprecipitation as previously reported(Hsu and Youle, supra, 1998). After incubating the sample with variousconcentrations such as 25, 50, 100, and 200 uM of FITC-labeled VPMLK(V5) peptide at 37° C. for 1 hour, immunoprecipitations were performedin detergent free condition at 37° C. by incubating 200 ul of lysateswith 20 ul of Protein G-Sepharose preabsorbed with 2 ug of anti-Baxantibody for 2 hours. FITC fluorescence of pre-immunoprecipitation(“B”+“F”) and the supernatant following immunoprecipitation (“F”) weremeasured by fluorescence microplate reader (Molecular Devices), and thevalues of “B+F” and “B” were calculated. Kd was calculated by Scatchardplot. For control experiment, immunodepletion of Bax was performedbefore the incubation of FITC-V5 and cell lysates. Two hundred ul ofcell lysates were mixed with 4 ug of anti-Bax antibody and incubated at37° C. for 2 hours for immunodepletion of Bax. No significant binding ofFITC-V5 to the cellular components was detected in Bax-immunodepletedsamples.

I claim:
 1. A method of protecting cells from cell death comprising thestep of supplying to the cell an effective amount of compositioncomprising a Bax-inhibiting peptide.
 2. The method of claim 1, whereinthe peptide comprises a peptide selected from the group consisting ofVPMLKE, VPMLK, PMLKE and PMLK.
 3. The method of claim 1 wherein thepeptide comprises VPMLKE.
 4. The method of claim 1 wherein the peptidecomprises VPMLK.
 5. The method of claim 1 wherein the peptide comprisesPMLKE.
 6. The method of claim 1 wherein the peptide comprises PMLK. 7.The method of claim 1 wherein the peptide is of the following formula:X¹PX²LX³X⁴, wherein X¹ is selected from amino acids with non-polar sidechain; X² is selected from amino acids with non-polar side chain; X³ isselected from amino acids with charged polar side chain; X⁴ is selectedfrom amino acids with charged polar side chain; and Either X¹ or X⁴ maybe absent.
 8. A method of protecting cells from cell death comprisingthe steps of: a) designing a peptide or chemical to mimic theBax-suppressing function of VPMLKE, VPMLK, PMLKE or PMLK, and b)supplying cells with an effective amount of the chemical or peptide. 9.An isolated peptide, wherein the peptide is VPMLKE.
 10. An isolatedpeptide, wherein the peptide is VPMLK.
 11. An isolated peptide, whereinthe peptide is PMLKE.
 12. An isolated peptide, wherein the peptide isPMLK.
 13. A pharmaceutical composition comprising a Bax-inhibitingpeptide and a pharmaceutical carrier, wherein the peptide is of thefollowing formula: X¹PX²LX³X⁴, wherein X¹ is selected for amino acidswith non-polar side chain; X² is selected for amino acids with non-polarside chain; X³ is selected for amino acids with charged polar sidechain; X⁴ is selected for amino acids with charged polar side chain; andEither X¹ or X⁴ may be absent.
 14. An isolated peptide wherein thepeptide is the peptide of claim
 13. 15. A method of preserving cells andorgans for transfusions or transplantation comprising storing the cellsor organs in an effective amount of the peptide of claim
 1. 16. A methodof regenerating damaged cells, comprising storing the cells in aneffective amount of the peptide of claim
 1. 17. A method of improvingtransfection efficiency of genes or proteins into cells, comprisingstoring the cells in an effective amount of the peptide of claim
 1. 18.The method of claim 1 wherein the Bax-inhibiting peptide is administeredto a patient.
 19. The method of claim 18 wherein the patient is a strokepatient.
 20. The method of claim 1 wherein the patient is a heart attackpatient.
 21. The method of claim 1 wherein the patient is an ischemiapatient.
 22. The method of claim 1 wherein the patient is a degenerativedisease patient.
 23. The method of claim 1 wherein the patient hasinfection caused by an agent selected from the group of bacteria,virus(es) and protozoa.
 24. The method of claim 1 wherein the patienthas drug side effects, wherein the drug is selected from the groupconsisting of anticancer drugs, and UV/X-ray irradiation.